ABSTRACT The rise of multi-drug resistant pathogens, particularly ESKAPE pathogens, is a major public health concern. One effective countermeasure against such pathogens is novel antibiotics, but the rate of antibiotic discovery has been in steady decline. One particularly promising source of novel antibiotics is microbial species because most of them have never been cultivated or explored. This is especially true about marine species, specifically microbes living in association with invertebrates: such symbioses are known producers of bioactive compounds, these compounds likely originate from associated microbes, but only few of them are available in culture. We have designed and developed several proprietary technology platforms to grow and isolate such ?uncultivable? microorganisms. These platforms are based on a simple idea: strains that do not grow in the lab can be grown in their natural environments if placed into small diffusion chambers. Cells inside such chambers, if returned back to the original environment, will be naturally fed by nutrients and growth factors diffusing through semipermeable membranes. This approach has resulted in the discovery of several new compounds, including teixobactin, an exciting new class of antibiotic that is active against resistant pathogens. This general approach can be used to grow invertebrate-associated, previously uncultivated microorganisms as well. For this project, we designed diffusion chambers that are flexible and can assume the topography of the animal, thus providing an immediate contact with the host ? and the compounds it produces. In preliminary studies, we validated this general approach by growing a diverse collection of novel microorganisms showing antimicrobial activities. Here we will capitalize on these advances and achieve the following Specific Aims. Aim 1. Developing a method to grow and isolate novel microbial species living in association with marine invertebrates. We will use our new method to isolate 5000 microorganisms from 4 invertebrate species. Aim 2. Screening for antimicrobial activity. We will ferment the strains from Aim 1 and screen them against a panel of Gram-negative and Gram-positive pathogens, prioritizing 100 strains for chemical dereplication. Aim 3. Chemical dereplication of antimicrobial compounds. Using traditional separation methodologies, bioassay-guided fractionation, and high-resolution mass spectrometry, we will identify extracts with novel antimicrobial compounds and isolate these compounds into pure substances. We aim at discovering 1-3 novel compounds with low toxicity and a good spectrum of activity and potency. The end products of this project, an innovative cultivation platform, a large collection of novel marine microorganisms, and a selection of antimicrobials they produce, will set the stage for development of novel antibiotics in the future Phase II application.